Ushijima Hitoshi, Yoshioka Hiroki, Esaki Ritsuko, Takahashi Keiji, Kuwayama Masashige, Nakane Takashi, Nagashima Hiroshi
Chiba Prefectural Animal Experimental Station, Japan.
J Reprod Dev. 2004 Aug;50(4):481-6. doi: 10.1262/jrd.50.481.
An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 microg/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +/- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +/- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.
采用三种具有不同冷却速率的玻璃化方法，研究了一种有效的猪桑椹胚冷冻保存方案（实验1）。还通过最小体积冷却（MVC）法检测了去除细胞质脂滴后玻璃化胚胎的存活率（实验2）。在实验1中，分别用0.25 ml塑料细管（ST法）、凝胶加样吸头（GLT法）和MVC法对桑椹胚期胚胎进行玻璃化处理，并储存在液氮中，之后在蔗糖溶液中复温，随后逐步去除冷冻保护剂。在实验2中，用7.5 μg/ml细胞松弛素B以12000×g离心桑椹胚20分钟，使细胞质脂滴极化，然后通过显微操作从胚胎中去除（去脂）。桑椹胚期去脂的胚胎和桑椹胚至囊胚期完整的胚胎均采用MVC法进行玻璃化处理。在两个实验中，通过在NCSU - 23 + 10% FCS中培养48小时来评估玻璃化胚胎的体外存活率。复温至囊胚后的玻璃化胚胎体外发育率，ST法为20%（6/30），GLT法为39%（18/46），MVC法为60%（26/43）。与完整玻璃化桑椹胚（24/42，57%，P<0.001）和囊胚（23/31，74%，P<0.05）相比，去脂后玻璃化处理可进一步提高胚胎存活率（95%，35/37）。此外，去脂玻璃化桑椹胚来源的囊胚中的细胞数（92±25）与完整对照未玻璃化胚胎来源的细胞数（103±31）相当。我们的结果表明，使用MVC法结合去脂处理时，玻璃化猪桑椹胚的存活率最高。
